The hepatic endoplasmic reticulum (ER)-anchored hemoproteins, cytochromes P450 (P450s), are instrumental in the metabolism of physiologically relevant endobiotics and xenobiotics (clinically relevant drugs, environmental toxins and carcinogens). In the course of this metabolism, however, the participating enzyme is sacrificed in a mechanism-based "suicide-inactivation". One form of this inactivation entails destruction of P450 prosthetic heme into fragments that irreversibly modify its protein at its active site. Such abnormal modification in vivo, triggers P450 degradation. The long-term goals of this proposal are centered on the hypothesis that heme-modification of the P450 protein predisposes it for degradation. Thus, they have centered on structural characterization of the heme-modified protein, and elucidation of the mechanism of its proteolytic degradation. Findings to date reveal that the heme-modified P450 3A4 protein is phosphorylated, ubiquitinated and degraded by the cytosolic 26S proteasome, raising some intriguing fundamental questions which are the subject of the present proposal: (i) How are the ER-bound P450 proteins delivered to the cytosolic proteasome? (ii) What precise sites (usually Lysepsilon-NH2) are required to be ubiquitinated for such P450 targeting to the 26S proteasome? (iii) Which particular ubiquitin conjugating enzymes are involved in this P450 targeting? And more importantly, is physiologically the native enzyme similarly disposed of as the heme-modified protein? While the ubiquitination and degradation are related, it is unclear whether phosphorylation is necessary for these events. Thus, the final aim (iv) is to determine the role of phosphorylation in P450 degradation by characterizing the cellular kinases involved, the protein sites phosphorylated and the use of selective inhibitors of the identified kinases as probes. Immunological, morphological, site-directed mutagenesis, biochemical, protein chemistry and mass spectrometric approaches will be used in elucidating these issues, using intact freshly isolated rat hepatocytes, COS-7 cells, and S. cerevisiae as models. The proposed studies center on a physiologically relevant but neglected aspect of P450 biology. Because P450s are integral ER-membrane proteins, elucidation of its turnover will provide a biological prototype for other ER-residents. Furthermore, these studies are focused on P450 3A4, the major human liver and intestinal enzyme and its rat orthologs, which are responsible for the metabolism of over 60 percent of clinically prescribed drugs, and are particularly susceptible to this biological fate. Finally, because the 26S proteasomal subunits are responsible for the generation of antigenic peptides, these studies may provide insight into the generation of P450 autoantibodies detected in sera of patients with chronic active and drug-induced hepatitis and hypersensitivity reactions.